Experimental and Toxicologic Pathology 68 (2016) 579–588

Contents lists available at ScienceDirect

Experimental and Toxicologic Pathology

journal homepage: www.elsevier.de/etp

Effect of genetically modified corn on the jejunal mucosa of adult male

albino rat
Marwa A.A. Ibrahim, MD* , Ebtsam F. Okasha
Histology Department, Faculty of Medicine, Tanta University, Egypt

A R T I C L E I N F O A B S T R A C T

Article history:
Received 1 May 2016 Genetically modified (GM) plants expressing insecticidal traits offer a new strategy for crop protection.
Received in revised form 3 September 2016 GM-corn contains Bacillus thuringiensis (Bt) genes producing delta endotoxins in the whole plant. Diet
Accepted 6 October 2016 can influence the characteristics of the gastrointestinal tract altering its function and structure. The aim
of this study was to evaluate the effect of GM-corn on the histological structure of jejunal mucosa of adult
Keywords: male albino rat using different histological, immunohistochemical and morphometrical methods.
Genetically modified corn Twenty adult male albino rats were divided into two equal groups; control and GM-corn fed group
Jejunal mucosa administered with 30% GM-corn for 90 days. Specimens from the jejunum were processed for light and
PCNA
electron microscopy. Immunohistochemical study was carried out using antibody against proliferating
Electron microscopy
cell nuclear antigen (PCNA). Different morphometrical parameters were assessed. Specimens from GM-
Rat
corn fed group showed different forms of structural changes. Focal destruction and loss of the villi leaving
denuded mucosal surface alternating with stratified areas were observed, while some crypts appeared
totally disrupted. Congested blood capillaries and focal infiltration with mononuclear cells were
detected. Significant upregulation of PCNA expression, increase in number of goblet cells and a significant
increase in both villous height and crypt depth were detected. Marked ultrastructural changes of some
enterocytes with focal loss of the microvillous border were observed. Some enterocytes had vacuolated
cytoplasm, swollen mitochondria with disrupted cristae and dilated rough endoplasmic reticulum (rER).
Some cells had dark irregular nuclei with abnormally clumped chromatin. It could be concluded that
consumption of GM-corn profoundly alters the jejunal histological structure.
ã 2016 Elsevier GmbH. All rights reserved.

1. Introduction MON810 corn and MON-40-3-2, RR soybean meal (Reichert et al.,

Genetically modified (GM) or transgenic crops have been grown
for human and animal consumption since the 1990s (James and
Krattiger, 1996). There are currently over 200 different GM-crops
with various traits approved for consumption in many countries
(Zdziarski et al., 2014). GM plants expressing insecticidal traits
offer a new strategy for crop protection, but at the same time,
present a challenge in terms of food safety assessment (Yanfang
et al., 2013), these plant products are becoming increasingly
common in the human food-chain, despite this, feeding studies
examining the effects of GM-crops on animal and human health
are relatively scarce (Snell et al., 2012). The most widespread GM-
plant materials with the highest importance at the feed market are
* Corresponding author at: Histology Department, Faculty of Medicine, Tanta
University, 31527, Tanta, Egypt.
E-mail addresses: maleox68@yahoo.com, maleox68@hotmail.com,
marwa.ibrahim@med.tanta.edu.eg (M.A.A. Ibrahim).
2012).
“MON810: Ajeeb YG” is a GM-corn that has resistance to borers,
and this variety was produced by incorporating the MON810,
produced by Monsanto company, in the Egyptian conventional
corn “Ajeeb” (Rayan et al., 2012). MON810 variety contains Cry1Ab
genes from Bacillus thuringiensis, and these genes produce delta
endotoxins in the whole plant. These endotoxins activate in the
alkaline environment of insects’ gut, and then the insects die
within 24–48 h (Tenuta et al., 2011).
Within the next few years, crops that have been genetically
engineered for Bacillus thuringiensis resistance could dramatically
lower production costs and provide farmers with new insect
control options (Ibrahim and Shawer, 2014). With respect to safety
of GM foods, there are conflicting opinions, some studies reported
that GM foods had potentially toxic properties, which could
provoke unintended effects of genetic modification and others
reported that it is safe for use (Tyshko et al., 2007; Abdo et al.,
2013).

http://dx.doi.org/10.1016/j.etp.2016.10.001
0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
580 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588
Diet can influence the characteristics of the gastrointestinal
tract since the intestinal mucous membrane is directly in contact
with food and absorbs the substances produced by digestion, also
digestive tract is the first site of contact for any ingested
compound. Furthermore, since the stomach and the intestines
are the sites of longest residence for any ingested product, these
should become the most important sites for the evaluation of an
ingested compound's toxicity (Montagne et al., 2003). In particular,
it has been reported that the diet may affect both small and large
intestine in terms of mucosal architecture, villous height and crypt
depth, epithelial cell proliferation and other features (Seralini et al.,
2007; Trabalza-Marinucci et al., 2008). Moreover, it is known that
diet and the histochemical characteristics of goblet cell mucins
and/or mucous membrane are strictly correlated (Morini and
Grandi, 2010).
Histomorphological changes have been widely used to assess
the effects of GM ingredients on the diets of mice and rats (Hartke
et al., 2005; Hedemann et al., 2006). So this work was performed to
study the effect of GM corn on the histological structure of jejunal
mucosa of adult male albino rat using different histological,
immunohistochemical and morphometrical methods.
2. Materials and methods
The present study was carried out on twenty adult male albino
rats, weighing 150–200 g. The animals were kept in adequate
ventilation and temperature, where food and water were
consumed freely throughout the experimental period. The
experiment was approved by the Local Ethics Committee of
Faculty of Medicine, Tanta University (Egypt). After a one-week
acclimatization period, animals were randomly divided into two
equal groups: Group I (Control group): received a diet of grain
conventional corn meal (non-GM) for 90 days. Group II received
GM-corn (Ajeeb YG; Bt MON810) obtained from the agricultural
administration, Sakha, Kafr Elsheikh governorate, Egypt. Flours
from GM-corn grains were formulated into the animals’ diet at a
concentration of 30% and administered for 90 days (El-Shamei
et al., 2012).
Animals were closely observed for their general health and
behavior. Animals’ feed consumption and total body weight were
recorded throughout the experiment. At the end of the experiment,
animals were anesthetized using intraperitoneal injection of
pentobarbital (40 mg/kg) (Gaertner et al., 2008). The jejunal
specimens were dissected, rinsed with phosphate buffered saline
(PBS) and prepared for light and electron microscopic examination.
2.1. For examination by light microscopy
Cross-sectioned jejunal specimens were fixed in 10% neutral
buffered formalin, washed, dehydrated, cleared and embedded in
paraffin. Sections of 5 mm thickness were stained with haematox-
ylin and eosin (H&E) for the study of general histological features
and Periodic Acid Schiff reagent (PAS) for detection of neutral
mucopolysaccharide (Bancroft and Gamble, 2008).
2.2. For immunostaining with proliferating cell nuclear antigen
(PCNA)
For detection of proliferating crypt cells, 5 mm thick sections
were dewaxed, rehydrated, and washed with phosphate buffered
saline (PBS) and then incubated with PBS containing 10% normal
goat serum. Sections were incubated with the mouse monoclonal
antibody PC10 against PCNA (sc-56, Santa Cruz Biotech, Santa Cruz,
USA) (1:100) overnight in a humid chamber at 4
C and then
incubated with biotinylated rabbit anti-mouse Ig (1:200) for
60 min at room temperature. Sections were incubated with a
streptavidin–biotin–horseradish peroxidase complex (1:100) pre-
pared 30 min in advance and mixed shortly before use with an
equal volume of PBS. The immunoreactivity was visualized using
3,30 -diaminobenzidine (DAB) hydrogen peroxide as a chromogen
and sections were counterstained with Mayer’s haematoxylin. The
negative control sections were prepared by excluding the primary
antibodies (Ramos-Vara et al., 2008).
2.3. For examination by transmission electron microscopy
Cross-sectioned jejunal specimens were divided into small
pieces and fixed in 4% phosphate buffered gluteraldehyde (0.1 M,
pH 7.3), post-fixed with 1% phosphate-buffered osmium tetroxide,
then dehydrated in ascending grades of ethanol. After immersion
in propylene oxide, the specimens were embedded in epoxy resin
mixture. Semithin sections (1 mm thick) were stained with 1%
toluidine blue and examined by light microscope for proper
orientation (Bozzola and Russell, 1999). Ultrathin sections
(80–90 nm) were stained with uranyl acetate and lead citrate, to
be examined by JEOL-JEM-100 transmission electron microscope
(Tokyo, Japan) at the Electron Microscopic Unit, Faculty of
Medicine, Tanta University, Egypt.
2.4. For examination by scanning electron microscopy
Longitudinal jejunal specimens were cut open to expose the
lumen, rinsed with phosphate buffered saline (PBS) and fixed in 4%
phosphate buffered gluteraldehyde (0.1 M, pH 7.4), then in
phosphate-buffered 1% osmium tetroxide, dehydrated in graded
alcohol series, put into amyl acetate, dried with liquid CO2 under
pressure with critical point dryer (E 3000) and coated with gold
particles (Rau et al., 2001). These samples were observed under
Jeol JSM scanning electron microscope (SEM), at the Electron
Microscopic Unit, Faculty of Medicine, Tanta University, Egypt.
2.5. Morphometric study
The images were acquired using a Leica microscope (DM3000,
Leica, Germany) coupled to a digital camera (DFC-290, leica,
Germany). The image analysis was done using Leica Qwin 500C
Image analyzer computer system (Leica Imaging System LTD.,
Cambridge, England) at Central Research Lab, Faculty of Medicine,
Tanta University, Egypt. Images were analyzed for:
2.5.1. The mean height of jejunal villi
The height of jejunal villi (from the tip of the villus to the villus-
crypt junction) were measured in H&E stained sections. Ten
randomly-selected non-overlapping microscopic fields for each
specimen were measured at a magnification power of 100.
2.5.2. The mean jejunal crypt depth
The crypt depth was measured in H&E stained sections. Ten
randomly-selected non-overlapping microscopic fields for each
specimen were measured at a magnification power of 100.
2.5.3. The mean number of goblet cells
Goblet cells in PAS-stained sections were counted in both villi
and crypts. Ten randomly-selected non-overlapping microscopic
fields for each specimen were measured at a magnification power
of 200.
2.5.4. The mean percentage of PCNA-immunopositive cells
This was calculated to quantitatively evaluate the number of
PCNA-positive immunostained nuclei at a magnification power of
400. The results were expressed as a percentage of the total
 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588
number of cells counted (number of labeled nuclei  100/total cell
number).
2.6. Statistical analysis
The data were analyzed by student t-test using statistical
package for social sciences statistical analysis software (version
11.5; SPSS Inc., Chicago, Illinois, USA). All values were expressed as
mean  standard deviation. Differences were regarded as signifi-
cant or highly significant if probability value P < 0.05 or P < 0.001
respectively (Dawson-Saunders and Trapp, 2001).
3. Results
No animals expressed any sign of ill health throughout the
experiment and no deaths were reported. There were no
observable alterations in the animals’ behavior, feed consumption
or average weight gain as there was a non-significant difference in
total body weights between the two dietary groups at the start and
at the end of the experiment (Initial weights in gm; control group
166.12  12.10, GM-corn fed group 167.65  12.42, and final
weights in gm; control group 251.83  13.31 GM-corn fed group
256.31 14.55).
3.1. Light microscopic results
3.1.1. Haematoxylin and eosin (H&E) stain
Examination of H&E-stained sections obtained from the control
group (group I) showed the normal histological features of the
jejunal mucosa with long finger like villi covered with simple
columnar epithelial cells (enterocytes) and goblet cells. The villi
had a core of loose connective tissue extending from the lamina
propria containing some connective tissue cells and lymphocytes.
Intestinal crypts of Lieberkuhn appeared as tubular invaginations
extending from the bases of villi into the lamina propria (Fig. 1a).
The enterocytes had eosinophilic cytoplasm and basal oval nuclei.
The luminal surface of the enterocytes was covered by a regular
continuous striated (brush) border. Goblet cells appeared as empty
spaces between the enterocytes (Fig. 1b).
In the GM-corn fed group (group II), the jejunal mucosa showed
different forms of structural changes such as distortion, shorten-
ing, flattening and fusion of some villi as well as deepening of the
crypts. In addition, shedding of some epithelial cells in the
intestinal lumen with subsequent erosion was observed in focal
areas, on the other hand, stratification of enterocytes and
numerous goblet cells were detected in other areas (Fig. 1c–f).
Enterocytes with vacuolated or clear cytoplasm and pyknotic
nuclei were observed at the basal aspect of the some villi (Fig. 1c
and d). Focal mononuclear cellar infiltration and congestion of
some blood capillaries were also detected (Fig. 1d and g). Moreover,
marked disruption, disorganization, distortion and shedding of
jejunal mucosa were observed at certain areas (Fig. 1h).
3.1.2. Periodic acid Schiff reagent (PAS) stain
Examination of the PAS-stained sections from the control group
revealed intact PAS-positive brush border of the villi and goblet
cells inbetween the epithelial cells lining jejunal mucosa (villi and
crypts) (Fig. 2a). On the other hand, sections from the GM corn-fed
group showed an apparent increase in the number of goblet cells
lining jejunal mucosa with an increased intensity of PAS reaction at
the brush border. Interruption of brush border at certain sites was
also observed (Fig. 2b).
3.1.3. PCNA immunohistochemical staining
Examination of PCNA-immunostained sections of the control
group showed that only a few number of immunopositive
581
epithelial cells lining the crypts expressed as brown nuclear
coloration (Fig. 3a). On the other hand, sections from GM-corn fed
group showed an apparent increase in the number of PCNA-
immunopositive epithelial cells lining the crypts as well as in the
inflammatory cells infiltrating the lamina propria (Fig. 3b and c).
3.2. Electron microscopic results
3.2.1. Transmission electron microscopy
Examination of ultrathin sections from control animals showed
that the enterocytes appeared regularly arranged and closely
packed, containing basal oval euchromatic nuclei with prominent
nucleoli, many mitochondria, and showed regular continuous
microvillous borders. Goblet cells were observed in between the
enterocytes with the characteristic mucin granules (Fig. 4a and b).
Ultrathin sections from the GM-fed group showed marked
ultrastructural changes in some enterocytes, with focal loss of the
microvillous border and partial shedding of some cells. Some
enterocytes contained vacuolated cytoplasm, swollen and degen-
erated mitochondria with disrupted cristae and dilated rough
endoplasmic reticulum (rER). Other cells contained dark irregular
nuclei with abnormally clumped chromatin (Fig. 4c–f). Goblet cells
expressed vacuolated cytoplasm and dilated rER (Fig. 4d and e).
3.2.2. Scanning electron microscopy
SEM examination of jejunum from the control animals revealed
the typical leaf-shaped villi of the rat small intestine (Fig. 5a).
Goblet cells distended with mucus were clearly observed in
between the enterocytes (Fig. 5b).
Examination of group II fed with GM-corn revealed apparent
erosions and fissures at the tips of the jejunal villi, they were
frequently surrounded by enterocytes of normal appearance
(Fig. 5c). In other areas, stratified cells were observed at the
villous surface together with some exfoliated sheets of cells
(Fig. 5d). A number of apically swollen cells clearly demarcated
from each other and several other cells which had lost their brush
border were observed (Fig. 5e). Occasional extravasations of red
blood cells were observed at certain sites of the villous surface
(Fig. 5f).
3.3. Morphometric and statistical analysis
There was a highly significant decrease in the mean height of
jejunal villi in the GM-corn fed group (482.09  9.31) compared to
the control group (507.01 10.10). Additionally, a highly significant
increase in the mean depth of jejunal crypts in the GM-corn fed
group (163.92  7.38) compared to the control group
(133.60  5.02) was detected (Table 1, Fig. 6a and b).
Moreover, the mean number of goblet cells counted in both villi
and crypts in the GM-fed group (76.38  3.22) showed a highly
significantly increase compared to the control (50.31  3.76).
Additionally, a highly significant increase (P < 0.001) in the number
of PCNA-immunopositive cells in the epithelial cells of the GM-
corn fed group (23.11  2.13) compared with the control group
(8.34  1.17) was detected (Table 1, Fig. 6c and d).
4. Discussion
Using genetically modified crops in human food or animal feed
raises a considerable amount of public concern about the possible
impact of GM crops on the environment, food safety and both
animal and human health, taking in consideration the importance
of ethical, political, and economical aspects. Conflicting reports
and contradicting opinions continuously rise about the possible
hazards of GM crops on human health in particular. In addition, too
little is known about the validity of the different safety
582 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588

Fig. 1. Light microscopic examination (H&E): a,b) control group showing long finger-like villi with a core of connective tissue (c) and covered with enterocytes (thin arrows)
and goblet cells (curved arrows), invaginated crypts (notched arrows) are between the bases of villi. The enterocytes have eosinophilic cytoplasm and basal oval nuclei. Notice
the regular striated border (arrow head) of the luminal surface of enterocytes. Some lymphocytes are seen (thick arrow). c–d) GM-corn fed group showing distortion (thick
arrow), shortening (thin arrow) and flattening of some villi (double arrows) with focal stratification of the enterocytes (arrow heads) and apparent increased number of goblet
cells (curved arrows), pyknotic cells are observed in the basal aspect of villous lining (angular arrows). Notice congested blood capillaries (V). e–f) GM-corn fed group showing
shortening (thin arrow) and fusion of some villi (double thick arrows), deepening of the crypts (notched arrows) and disruption of the villous covering epithelium with the
 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588 583

Fig. 2. PAS staining: a) control group showing PAS positive reaction in goblet cells (thin arrows) and intact brush border of jejunal mucosa (thick arrow). b) GM-corn fed group
showing numerous goblet cells with strong positive reaction (thin arrows). Notice focal interruption of the brush border (thick arrow). [Magnification 400, scale
bar = 50 mm].

Fig. 3. PCNA-immunostaining a) control group showing some PCNA immunostained positive cells lining the crypts expressing brown coloration (thin arrows). b, c) GM-corn
fed group showing numerous PCNA immunostained positive cells lining the crypts (thin arrows) as well as in the inflammatory cells infiltrating the lamina propria (thick
arrows). [Magnification 400, scale bar = 50 mm].

assessments tests that were undertaken for these crops. The need
for deeper insight into the influence of one of the most common
GM crops in Egypt (MON810: Ajeeb YG) has encouraged the design
of this work to assess the impact of its long term consumption on
the jejunal mucosa of adult male albino rat employing different
histological, immunohistochemical and morphomterical techni-
ques.
In the current work, focal structural changes including
distortion, shortening, flattening and fusion of some jejunal villi
were observed in GM-corn fed group. In addition, stratification
alternating with shedding of the jejunal surface epithelium were
detected as was similarly reported (Fares & El-Sayed 1998). These
changes could be observed as early as after only 45 days of GM-
corn consumption (El-Shamei et al., 2012). Moreover, some
researchers have suggested that significant GM-maize linked
effects were generally detected either after 14 weeks of
consumption or at a high GM feed dose in the diet (de Vendômois
et al., 2009). Additionally, stratification and shedding of epithelium
of the villi in this study came in association with a significantly
increased crypt proliferation as detected using immunohisto-
chemical staining against PCNA. Some authors provided evidence
of proliferative activation of basal epithelial cells of alimentary
canal in all GM-corn fed animals indicating an increased turnover
rate (Trabalza-Marinucci et al., 2008).
Erosions in the villi and denuded mucosal surface were also
evident as observed on both light and electron microscopic levels
in GM-corn fed group. Erosion is a very alarming finding as it could
lead to occasional hemorrhage especially in more vulnerable
groups. Similar erosions were observed in rat jejunum upon
chronic intake of dietary fibers (Cassidy et al., 1981). They observed
as well large denuded areas where the cell demarcations were very
evident. Although they suggested a possible correlation with bile
acids sequestration, they did not give much of an explanation for
such changes. Yet, it could be suggested that these surface erosions
are most likely attributed to the inflammatory changes observed
during the current study, where inflammatory signs in the jejunum
of GM-corn fed group were detected in the form of dilated
congested blood vessels and mononuclear cellular infiltration in
the lamina propria, in addition to cellular and hemorrhagic debris
observed with the scanning electron microscopy. This coincides
with the observations of a previous work, where a higher rate of
severe inflammation in the stomach of pigs upon mixed GM maize
and soyabean feed consumption was documented (Carman et al.,
2013). One explanation for the inflammation could be the action of

enterocytes shed into the lumen (wavy arrow) leaving eroded villous surface (thick arrow). Notice focal stratification of the enterocytes (arrow heads). g–h) GM-corn fed
group showing area of focal mononuclear cellular infiltration in lamina propria (thick arrow). Notice areas of disrupted mucosa (thin arrows) with sloughing of the villi
(double arrows) and the crypts (notched arrows) leaving denuded areas (arrow head). [Magnification; (a, c, e, g and h) 200, scale bar = 100 mm (b, d and f) 400, scale
bar = 50 mm].
584 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588

Fig. 4. Transmission electron microscopy: a, b) control group showing regularly arranged closely packed enterocytes with basal oval euchromatic nuclei (N) and prominent
nucleolus (n), mitochondria (M), rough endoplasmic reticulum (R) and an intact microvillous border (arrow). Notice goblet cell with mucin granules (g). [Magnification 8780
and 11,700 respectively, scale bar = 2 mm]. c–f) GM-corn fed group showing enterocytes containing nuclei with abnormally clumping chromatin (N), swollen degenerated
mitochondria with disrupted cristae (M), rarefaction and vacoulation of the cytoplasm (v), dilated rER (R) and focal loss of the microvillous border (arrow) with loss of the
apical part of the enterocyte (asterisk). Notice goblet cell with coalesced mucin granules (g) and dilated rER (R). [Magnification (c,d and e) 11,700, scale bar = 2 mm and f
29200, scale bar = 500 nm].
 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588 585

Fig. 5. Scanning electron microscopy: a, b) control group showing the typical leaf-shaped villi with smooth microvillous surface (thick arrows) and goblet cells distended with
mucus (wavy arrow) inbetween the enterocytes (arrow head). c,d) GM-corn fed group showing apparent erosions (thin arrows), fissures (double thin arrows) at the tips of the
jejunal villi and areas of apparent stratification (notched arrows) and exfoliated sheets of cells (angular arrows). Notice surrounding enterocytes of normal appearance (arrow
head) and distorted and damaged cells (curved arrow). e,f) GM-corn fed group showing number of apically swollen cells clearly demarcated from each other (thin arrows) and
several other cells which had lost their brush border (notched arrows) with areas of enterocytes of normal appearance inbetween (arrow head). Extravasations of red blood
cells are observed at certain sites of the villous surface (asterisks). Notice distorted and damaged cells (curved arrow). [Magnification (a, c and d) 150, scale bar = 100 mm, (b,
e and f) 1000, scale bar = 10 mm].
586 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588
Table 1
Morphometric analysis of jejunal specimens of all groups.
Parameters
Mean height of jejunal villi (mm)
Mean depth of jejunal crypts (mm)
Mean number of goblet cells
Mean% of PCNA immunopositive cells
Data are expressed as mean  standard deviation, * indicates significance.
Cry 1Ab proteins produced by GM-corn that act as insecticides by
inducing pore formation and disintegration of the gut tissue of
certain borers that attack the corn plants (Fiuza et al., 2013).
Additionally, some authors have collectively attributed the
histopathological and biochemical changes observed upon GM-
corn consumption to the possible role of the delta-endotoxin
produced by the Bt corn (Abdo et al., 2014). In support of this
hypothesis, six proteins in the mouse small intestine that could
bind to a Cry protein (Cry 1Ac) were recognized (Vazquez-Padron
et al., 2000), they added that when the Cry protein binds to these
proteins, it results in hyperpolarisation of the intestine, which is
consistent with the formation of cationic channels, as occurs in the
insect gut. Moreover, persistence of Cry 1Ab proteins throughout
the digestive tract of pigs was detected, indicating the resistance of
these proteins to digestion (Chowdhury et al., 2003; Walsh et al.,
2012b). On the contrary, Cry1Ab protein was indicated to bind at
low levels to the cytoskeletal protein actin, which is a structural
protein, but not to extracellular proteins that have been identified
as receptors for Cry protein binding on target insect mid
gastrointestinal tract epithelia (Shimada et al., 2006).
Fig. 6. Morphometrical and statistical analysis a) mean height of jejunal villi b) mean depth of jejunal crypts.c) mean number of goblet cells d) mean percentage of PCNA-
immunopositive cells. *P < 0.05 is significant versus control, n = 10 in each group.
Control group GM-corn fed group P value
507.01  10.10 482.09  9.31* P < 0.001
133.60  5.02 163.92  7.38* P < 0.001
50.31  3.76 76.38  3.22* P < 0.001
8.34  1.17 23.11  2.13* P < 0.001
In the current study, several morphometrical parameters were
assessed. Mean number of goblet cells was found to be significantly
higher in GM-corn fed group compared to the control group. This
came in accordance with the work of others studying 30% GM corn
consumption in male albino rats after 45 days (El-Shamei et al.,
2012). Goblet cells synthesize and secrete mucous layer that covers
the gastrointestinal epithelium, providing protection against
pathogens, lubrication for intestinal content and a medium to
transport nutrients across the intestinal lumen to the epithelial
cells. The mucous layer is composed of mucins that are secreted at
a baseline rate, but in case of insult, bioactive compounds
stimulates the goblet cells to increase mucin secretion either
directly or by stimulating host cytokines such as TNF-alpha and IL-
6 (Deplancke and Gaskins, 2001). Nevertheless, changes in the
intestinal mucins are directly linked to the dynamic equilibrium
between their biosynthesis by goblet cells and their degradation
within the lumen by the microflora, thus the levels of mucins may
vary in the epithelium according to the diet and to bacteria
inhabiting the digestive tract (Montagne et al., 2003).
 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588
Similarly, an increase in the number of goblet cells/mm in
jejunal villi was previously reported upon studying the effect of
38.9% of the very same GM-corn on male weanling pigs for 31 days
(Walsh et al., 2012a), they attributed it to a possible change in gut
microflora that enhanced inflammation and decreased integrity of
the mucosal barrier. Intestinal commensal microflora have been
reported to directly impact the intestinal epithelial functions
including those of goblet cells (Kim and Ho, 2010) by producing
mucin-degrading enzymes (Macfarlane et al., 2005) or by
stimulation of mucin gene expression (Dohrman et al., 1998).
On the other hand, a significant decrease in mean villous height
coupled with a significant increase in mean crypt depth was
recorded during the current study. This could be indicative of the
high enterocytes proliferation in the crypts and high exfoliation in
the villi as similarly reported in response to different microflora in
intestine (Shirkey et al., 2006). On the contrary, others reported a
decrease in the villous height but did not comment on the crypt
depth (El-Shamei et al., 2012), while an increase in both
parameters throughout the small intestine could be detected by
Walsh et al. (2012a). The discrepancies between different research
works could be in part attributed to the variable doses of GM feed
used in the different studies ranging between 11% and 38.9%, while
the consumption durations ranged between 14 days and 3 years.
Transmission electron microscopic examination of specimens
from the GM-corn fed group revealed marked ultrastructural
changes mainly including nuclear irregularities with abnormal
chromatin clumping, dilated rER and distorted mitochondria.
Similarly, smaller cell nuclei containing increased amounts of
heterochromatin were reported in the liver and pancreas of GM
corn-fed animals (Trabalza-Marinucci et al., 2008). Moreover,
nuclear alterations, mainly small, irregular and abnormal chroma-
tin clumping in mice hepatocytes upon 90 days of GM soyabean
consumption were as well recorded (Malatesta et al., 2005), they
attributed these observations to a GM-induced modification in the
metabolic activity of the cells although that the underlying
mechanism remains elusive. Nevertheless, degenerated mitochon-
dria and rER in association with delta endotoxin-treated mice were
typically described (Fares and El-Sayed, 1998). Fine structural
modifications of cellular components in relation to GM feed intake
have already been described, yet, reports have assumed that there
were no consequences on organ functions or animal health
(Malatesta et al., 2002).
In summary, the current work presents a multi-approach
histological assessment of a typical 90-days subchronic toxicity
study through combining different histological, histochemical,
immunohitochemical and morphometrical methods supported
with both transmission and scanning electron microscopy for a full
histological evaluation of the influence of GM-corn on the jejunum
in a rat model. Results from the current study could show that in
spite of the assuring reports on GM products, GM-corn has
profoundly altered the histological structure of the jejunal mucosa
at many levels and revealed several alarming signs as the
proliferative and eroded hemorrhagic lesions in addition to several
ultrastructural alterations described here for the first time for
jejunum under GM-corn influence. Possible mechanisms have
been proposed including inflammation associated with goblet cell
overexpression and PCNA upregulation. This should motivate the
conduction of a more extensive research to reveal the exact
mechanism of such unintended effect and how to remodulate the
GM crops to avoid their adverse effects.
Conflict of interest
The authors have no conflict of interest to declare.
587
References
Abdo EM, Barbary OM, Shaltout OE. Chemical Analysis of BT corn Mon-810: Ajeeb-
YG1 and its counterpart non-Bt corn “Ajeeb”. IOSR J. Appl. Chem. (IOSR-JAC)
2013;4(1):55–60.
Abdo EM, Barbary OM, Shaltout OE. Feeding study with bt corn (MON810: Ajeeb YG)
on rats: biochemical analysis and liver histopathology. Food Nutr. Sci.
2014;5:185–95.
Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. sixth ed.
Philadelphia: Churchill Livingstone: Elsevier; 2008. p. 126–7.
Bozzola JJ, Russell LD. Electron Microscopy Principles and Techniques for Biologists.
2nd ed. Toronto, London: Jones and Bartlett Publishers; 1999. p. 16.
Carman JA, Vlieger HR, Ver Steeg LJ, Sneller VE, Robinson GW, Clinch-Jones CA,
Haynes JI, Edwards JW. A long-term toxicology study on pigs fed a combined
genetically modified (GM) soy and GM maize diet. J. Org. Syst. 2013;8(1):38–54.
Cassidy MM, Lightfoot FG, Grau LE, Story JA, Kritchevsky D, Vahouny GV. Effect of
chronic intake of dietary fibers on the ultrastructural topography of rat jejunum
and colon: a scanning electron microscopy study. Am. J. Clin. Nutr. 1981;34:218–
28.
Chowdhury EH, Kuribara H, Hino A, Sultana P, Mikami O, Shimada N, Guruge KS,
Saito M, Nakajima Y. Detection of corn intrinsic and recombinant DNA
fragments and Cry1Ab protein in the gastrointestinal contents of pigs fed
genetically modified corn Bt11. J. Anim. Sci. 2003;81:2546–51.
Dawson-Saunders B, Trapp R. Basic and Clinical Biostatistics. third ed. Lange
Medical Book/McGraw-Hill Medical Publishing Division; 2001. p. 161–218.
Deplancke B, Gaskins HR. Microbial modulation of innate defense: goblet cells and
the intestinal mucus layer. Am. J. Clin. Nutr. 2001;73(6):1131S–4S.
Dohrman A, Miyata S, Gallup M, Li J, Chaelin C, Coste A, Escudier E, Nadel J, Basbaum
C. Mucin gene (MUC 2 and MUC 5AC) upregulation by gram-positive and gram-
negative bacteria. Biochim. Biophys. Acta 1998;1406:251–9.
El-Shamei ZS, Gab-Alla AA, Shatta AA, Moussa EA, Rayan AM. Histopathological
changes in some organs of male rats fed on genetically modified corn (Ajeeb
YG). J. Am. Sci. 2012;8(10):684–96.
Fares NH, El-Sayed AK. Fine structural changes in the ileum of mice fed on delta-
endotoxin-treated potatoes and transgenic potatoes. Nat. Toxins 1998;6
(6):219–33.
Fiuza LM, Knaak N, Pires da Silva RF, Henriqu JAP. Receptors and lethal effect of
bacillus thuringiensis insecticidal crystal proteins to the anticarsia gemmatalis
(Lepidoptera, Noctuidae). ISRN Microbiol. 2013; (Article ID: 940284).
Gaertner DJ, Hallman TM, Hankenson FC, Batchelder MA. Anesthesia and analgesia
for laboratory rodents. In: Fish RE, Danneman PJ, Brown m, Karas AZ, editors.
Anesthesia and Analgesia in Laboratory Animals. 2nd ed. Academic Press:
London (UK); 2008. p. 239–97.
Hartke JL, Monaco MK, Wheeler MB, Donovan SM. Effect of a short-term fast
intestinal disaccharidase activity and villus morphology of piglets suckling
insulinlike growth factor-I transgenic sows. J. Anim. Sci. 2005;83:2404–13.
Hedemann MS, Eskildsen M, Laerke HN, Pedersen C, Lindberg JE, Laurinen P,
Knudsen KE. Intestinal morphology and enzymatic activity in newly weaned
pigs fed contrasting fiber concentrations and fiber properties. J. Anim. Sci.
2006;84:1375–86.
Ibrahim RA, Shawer DM. Transgenic Bt-plants and the future of crop protection (an
overview). Int. J. Agric. Food Res. 2014;3(1):14–40.
James C, Krattiger AF. Global Review of the Field Testing and Commercialization of
Transgenic Plants, 1986 to 1995: the First Decade of Crop Biotechnology. Ithaca,
NY: ISAAA Briefs No. 1. ISAAA; 1996. p. 31.
Kim YS, Ho SB. Intestinal goblet cells and mucins in health and disease: recent
insights and progress. Curr. Gastroenterol. Rep. 2010;12:319–30.
Macfarlane S, Woodmansey EJ, Macfarlane GT. Colonization of mucin by human
intestinal bacteria and establishment of biofilm communities in a two stage
continuous culture system. Appl. Environ. Microbiol. 2005;71:7483–92.
Malatesta M, Caporaloni C, Gavaudan S, Rocchi MBL, Tiberi C, Gazzanelli G.
Ultrastructural morphometrical and immunocytochemical analyses of
hepatocyte nuclei from mice fed on genetically modified soybean. Cell. Struct.
Funct. 2002;27:173–80.
Malatesta M, Baldelli B, Battistelli S, Tiberi C, Manuali E, Biggiogera M. Reversibility
of hepatocyte nuclear modifications in mice fed on genetically modified
soybean. Eur. J. Histochem. 2005;4(3):237–42.
Montagne L, Pluske JR, Hampson DJ. A review of interactions between dietary fibre
and the intestinal mucosa, and their consequences on digestive health in young
non-ruminant animals. Anim. Feed. Sci. Technol. 2003;108(1–4):95–117.
Morini G, Grandi D. Methods to measure gastric mucosal lesions in the rat. Curr.
Protoc. Toxicol. 2010;43: (21.2. 1-21.2.15).
Ramos-Vara JA, Kiupel M, Baszler T, Bliven L, Brodersen B, Chelack B, West K, Czub S,
Del Piero F, Dial S, Ehrhart EJ, Graham T, Manning L, Paulsen D, Valli VE.
Suggested guidelines for immunohistochemical techniques in veterinary
diagnostic laboratories. J. Vet. Diagn. Invest. 2008;20:393–413.
Rau EI, Gostev AV, Zhu Shiqiu D, Phang D, Chan D, Thong D, Wong W. Comparative
analysis of scanning electron microscopy techniques for semiconductors:
electron-beam-induced potential method, single-contact electron-beam-
induced current method, and thermoacoustic detection. Russ. Microlectron.
2001;30(4):207–18.
Rayan AM, Gab-Alla AA, El-Shamei ZS, Shatta AA, Moussa EA. Morphological and
biochemical changes in male rats fed on genetically modified corn (Ajeeb YG). J.
Am. Sci. 2012;8(9):1117–23.
588 M.A.A. Ibrahim, E.F. Okasha / Experimental and Toxicologic Pathology 68 (2016) 579–588
Reichert M, Kozaczyn
ski W, Karpin
˛tkiewicz

ska TA, Bocian Ł, Jasik A, Kycko A, Swia

˛tkiewicz S, Furgał-Dierzuk
M, Swia _ I, Arczewska-Włosek A, Strzetelski J, Kwiatek
K. Histopathology of internal organs of farm animals fed genetically modified
corn and soybean meal. Bull. Vet. Inst. Pulawy 2012;56:617–22.
Seralini GE, Cellier D, de Vendomois JS. New analysis of a rat feeding study with a
genetically modified maize reveals signs of hepatorenal toxicity. Arch. Environ.
Contam. Toxicol. 2007;52(4):596–602.
Shimada N, Miyamoto K, Kanda K, Murata H. Bacillus thuringiensis insecticidal
Cry1Ab toxin does not affect the membrane integrity of the mammalian
intestinal epithelial cells: an in vitro study. In Vitro Cell. Dev. Biol. Anim.
2006;42:45–9.
Shirkey TW, Siggers RH, Goldade BG, Marshall JK, Drew MD, Laarveld B, Van kessel
AG. Effects of commensal bacteria on intestinal morphology and expression of
proinflammatory cytokines in the gnotobiotic pig. Exp. Biol. Med.
2006;231:1333–45.
Snell C, Bernheim A, Berge J, Kuntz M, Pascal G, Paris A, Ricroch AE. Assessment of
the health impact of GM plant diets in long-term and multigenerational animal
feeding trials: a literature review. Food Chem. Toxicol. 2012;50(3–4):1134–48.
Tenuta A, Sears M, Meloche F, Schaafsma A. A Grower’s Handbook Controlling
European Corn Borer with BT Corn Technology. . p. 1–16.
Trabalza-Marinucci M, Brandi G, Rondini C, Avellini L, Giammarini C, Costarelli S,
Acuti G, Orlandi C, Filippini G, Chiaradia E, Malatesta M, Crotti S, Antonini C,
Amagliani G, Manuali E, Mastrogiacomo AR, Moscati L, Haouet MN, Gaiti A,
Magnani MA. Three-year longitudinal study on the effects of a diet containing
genetically modified Bt176 maize on the health status and performance of
sheep. Livest. Sci. 2008;113:178–90.
Tyshko NV, Aksyuk IN, Tutelyan VA. Safety assessment of genetically modified
organisms of plant origin in the Russian Federation. Biotechnol. J. 2007;2
(7):826–32.
Vazquez-Padron RI, Gonzales-Cabrera J, Garcia-Tovar C, Neri-Bazan L, Lopez-Revilla
R, Hernandez M, Moreno-Fierro L, de la Riva GA. Cry1Ac protoxin from Bacillus
thuringensis sp. kurstaki HD73 binds to surface proteins in the mouse small
intestine. Biochem. Biophys. Res. Commun. 2000;271:54–8.
Walsh MC, Buzoianu SG, Rea MC, O’Donovan O, Gelencser E, Ujhelyi G, Ross RP,
Gardiner GE, Lawlor PG. Effects of feeding Bt MON810 maize to pigs for 110 days
on peripheral immune response and digestive fate of the cry1Ab gene and
truncated Bt toxin. PLoS One 2012a;7:e36141.
Walsh MC, Buzoianu SG, Gardiner GE, Rea MC, Ross RP, Cassidy JP, Lawlor PG. Effects
of short-term feeding of Bt MON810 maize on growth performance, organ
morphology and function in pigs. Br. J. Nutr. 2012b;107:364–71.
Yanfang Y, Wentao X, Xiaoyun H, Haiyan L, Sishuo C, Xiaozhe Q, Kunlun H, Yunbo L.
Effects of genetically modifiedrice on the GI health of rats after 90-day
supplement. Sci. Rep. 2013;3:1962.
Zdziarski IM, Edwards JW, Carmanb JA, Haynes JI. GM crops and the rat digestive
tract: a critical review. Environ. Int. 2014;73:42.
de Vendômois JS, Roullier F, Cellier D, Séralini G. A comparison of the effects of three
GM corn varieties on mammalian health. Int. J. Biol. Sci. 2009;5(7):706–26.